The present invention relates to the field of infectious diseases. It finds particular application as a method of evaluating the response of Prions (Proteinaceous Infectious Agents) to various treatments, and will be described with particular reference thereto. It should be appreciated, however, that the invention is also applicable to other studies of prion activity.
The term “Prion” is used to describe proteinaceous-infectious agents that cause relatively similar brain diseases in humans and/or in animals, which are invariably fatal. These diseases are generally referred to as transmissible spongiform encephalopathies (TSEs). TSEs include Creutzfeldt-Jakob disease (CJD) and variant CJD (vCJD) in humans, Bovine Spongiform Encephalopathy (BSE) in cattle, also know as “Mad Cow Disease,” Scrapie in sheep, and Wasting Disease in elk. All of these diseases attack the neurological organs of the animal or animals which are susceptible to the particular disease. They are characterized by initially long incubation times followed by a short period of neurological symptoms, including dementia and loss of coordination, and eventually death.
The infectious agent responsible for these diseases is thought to be a simple protein, with no associated nucleic acids. The pathogenic mechanism for such prion diseases is proposed to involve an initially normal host encoded protein. The protein undergoes a conformational change to an abnormal form (a prion), which has the ability of self-propagation. The exact cause of this change is, at present, unknown. The abnormal form of the protein is not broken down effectively in the body and its accumulation in certain tissues (in particular neural tissue) eventually causes tissue damage, such as cell death. Once significant neural tissue damage has occurred, the clinical signs are observed.
Prion diseases may thus be classified as protein aggregation diseases, which also include several other fatal diseases, such as Alzheimer's disease and amyloidosis. In the case of CJD, the most prevalent prion disease in humans (occurring in roughly 1:1,000,000 of the population), about 85% of cases are thought to arise sporadically, about 10% are thought to be inherited, and about 5% arise iatrogenically.
There are currently no known effective treatments for prion diseases in animals or humans, and death thus follows the onset of neurological symptoms. Progress in the identification of target treatment drugs has been slow, due to the inability to perform testing in vitro. To date, no methods for culturing prions in media in the laboratory have been developed. In vivo studies involve inoculating a test animal with prions and examining the animal's response to a proposed treatment regime. Because progress of the disease is slow, these in vivo studies are inevitably lengthy and are thus not readily amenable to the screening of large numbers of potential drugs. In vivo mouse or hamster models have been engineered to be more susceptible to prions and are generally used for evaluations. In addition, because these diseases tend to be animal specific, it is not known whether tests done on animals can be readily applied to humans.
Some research groups have suggested using a yeast prion model for drug evaluation and there has been some reports of an in vitro model to study prion folding. However, there have been no studies which have established correlations between the behavior of these proposed models and prion activity.
In the early 1980's, a novel replicating agent was isolated from the human intestinal tract. (Burdon, J. Med. Micro., 29: 145–157 (1989)). This agent was isolated from the ileostomy fluid (filtered through a 0.2μ filter) of two patients with Crohn's disease, and could be cultured in vitro. It was given the name Ileal Fluid Dependent Organism (IFDO), although it has been subsequently found to survive in other media, such as in the presence of pancreatin. Discrete brown colonies were observed on a specific, select growth media. On examination of this agent, it did not appear to be viral, bacterial, or fungal in nature, but did appear to grow logarithmically and have unusual resistance to a variety of antibiotics, and physical and chemical agents. The agent was also found to have a high resistance to moist heat. This agent has not previously been directly linked to prions or used in prion research.
Although prion diseases have not generally been considered to be highly contagious, they can be transmitted within a species and, under certain conditions, from one species to another. It has recently been shown that prion diseases may be transmitted via high risk tissues, including the brain, spinal cord, and eye. Iatrogenic transmission has also been reported, including transmission via dura mater grafting, corneal transplants, pericardial homografts, human gonadotropin, and human growth hormone contamination. Transmission via medical devices has also been reported, including through reuse of neurosurgical instruments, depth electrodes, and other devices used during surgeries in close proximity to the central nervous system.
There is currently much speculation about the efficacy of conventional decontamination and sterilization methods for destruction of prions. Prions are notoriously very hardy and demonstrate resistance to routine methods of decontamination and sterilization. Some recommended methods include incineration, prolonged steam autoclaving, sodium hydroxide and sodium hypochlorite treatments at high concentrations (e.g., 1M NaOH or NaHClO3 at 2% available Cl for 1 hr.). These aggressive treatments are often incompatible with medical devices, particularly flexible endoscopes and other devices with plastic, brass, or aluminum parts. Many devices are damaged by exposure to high temperatures. Chemical treatments, such as strong alkali, are damaging to medical device materials or surfaces in general. Glutaraldehyde, formaldehyde, hydrogen peroxide, most phenolics, alcohols, and processes such as dry heat, boiling, freezing, UV, ionizing, and microwave radiation have generally been reported to be ineffective. There is a clear need for products and processes that are effective against prions yet compatible with surfaces.
One less aggressive treatment which has been investigated and shown to be effective against prions is a peracetic acid formulation formulated by STERIS Corporation, Mentor, Ohio, under the tradename STERIS 20™. The formulation contains peracetic acid in a blend of buffers, anticorrosives, surfactants, and chelators, prepared in a use dilution for sterile processing at above room temperature.
However, there is currently no ready means of evaluating anti-prion (“priocidal”) treatments. Culturing prion-treated devices after proposed priocidal treatments involves inoculating animals with washings from the devices and observing the development of the disease if the priocidal treatment is ineffective. This is a lengthy process and prone to errors, since the numbers of prions remaining on the devices may be relatively small. Additionally, there is a risk that prions which are not destroyed by the priocidal treatment may pose hazards to workers.
There are thus increased concerns among medical personnel regarding the proper care of patients identified as having prion diseases. There are also concerns that the diseases may be transmitted, through reuse of instruments and the like, due to a failure to detect the disease state prior to death of the infected patient. Additionally, the risks associated with high, medium, and low risk tissues have not yet been established. For example, tonsillectomy and dental procedures have been considered to be low risk procedures for potential prion infection. However, recent evidence suggests the risks may higher, due to the finding that prion infected tissues are being found outside the brain. It has also been suggested that there may be a link between prion-related diseases and similar disease states, such as Parkinson's and Alzheimer's diseases.
The present invention provides a new and improved method for evaluation of priocidal activity, which overcomes the above-referenced problems, and others.